Classical Serotypes Research Articles

Development and validation of an ELISA kit for the detection of Staphylococcus aureus enterotoxin A, B, C1, C2, C3, D, E from food samples

Staphylococcus aureus (S. aureus) is a significant food-borne pathogen that poses a threat to human health. The classical serotypes staphylococcal enterotoxin (SE) A, B, C1, C2, C3, D, E produced by S. aureus are the primary cause of its food-borne pathogenic toxicity. Therefore, establishing a highly sensitive, rapid, robust, high-throughput detection method for the total amount of classical serotypes of SEs is crucial for food safety and public health. This study prepared 10 rabbit polyclonal antibodies and 40 mouse monoclonal antibodies targeting SEA, SEB, SEC1, SEC2, SEC3. Upon identifying the optimal antibody pairs through square array method and optimization, a sandwich ELISA kit for the detection of SEs (SEA, SEB, SEC1, SEC2, SEC3, SED, SEE) was developed, with respective limits of detection at 0.169–0.688 ng/mL. In addition, this sandwich ELISA kit could detect SEs in raw cow milk, pasteurized milk, fermented milk, milk powder, whey powder, rice, raw pork, and canned beef samples with coefficients of variation below 7%. Meanwhile, this sandwich ELISA kit was stable at 4 °C for one-year storage. The study fills a research gap in the detection of the total amount of classical serotypes of SEs.

Rapid discrimination of four Salmonella enterica serovars: A performance comparison between benchtop and handheld Raman spectrometers.

Foodborne illnesses, particularly those caused by Salmonella enterica with its extensive array of over 2600 serovars, present a significant public health challenge. Therefore, prompt and precise identification of S. enterica serovars is essential for clinical relevance, which facilitates the understanding of S. enterica transmission routes and the determination of outbreak sources. Classical serotyping methods via molecular subtyping and genomic markers currently suffer from various limitations, such as labour intensiveness, time consumption, etc. Therefore, there is a pressing need to develop new diagnostic techniques. Surface-enhanced Raman spectroscopy (SERS) is a non-invasive diagnostic technique that can generate Raman spectra, based on which rapid and accurate discrimination of bacterial pathogens could be achieved. To generate SERS spectra, a Raman spectrometer is needed to detect and collect signals, which are divided into two types: the expensive benchtop spectrometer and the inexpensive handheld spectrometer. In this study, we compared the performance of two Raman spectrometers to discriminate four closely associated S. enterica serovars, that is, S. enterica subsp. enterica serovar dublin, enteritidis, typhi and typhimurium. Six machine learning algorithms were applied to analyse these SERS spectra. The support vector machine (SVM) model showed the highest accuracy for both handheld (99.97%) and benchtop (99.38%) Raman spectrometers. This study demonstrated that handheld Raman spectrometers achieved similar prediction accuracy as benchtop spectrometers when combined with machine learning models, providing an effective solution for rapid, accurate and cost-effective identification of closely associated S. enterica serovars.

Fourier transform infrared spectroscopy for Streptococcus pneumoniae capsular serotype classification in pediatric patients with invasive infections.

Invasive pneumococcal disease (IPD) is a major cause of morbidity and mortality worldwide, particularly in the pediatric population (children and infants), with high rates of hospitalization and death. This study aimed to create and validate a classifier for Streptococcus pneumoniae serotyping using Fourier-transform infrared (FT-IR) spectroscopy as a rapid alternative to the classical serotyping technique. In this study, a database comprising 76 clinical isolates, including 18 serotypes (predominantly serotypes 19A, 6C, and 3) of S. pneumoniae from pediatric patients with IPD, was tested at a tertiary pediatric hospital in southern Brazil during 2016-2023. All isolates were previously serotyped using the Quellung reaction, and 843 FT-IR spectra were obtained to create a classification model using artificial neural network (ANN) machine learning. After the creation of this classifier, internal validation was performed using 384 spectra as the training dataset and 459 as the testing dataset, resulting in a predictive accuracy of 98% for serotypes 19A, 6, 3, 14, 18C, 22F, 23A, 23B, 33F, 35B, and 9N. In this dataset, serotypes 10A/16F, 15ABC, and 7CF could not be differentiated and were, therefore, grouped as labels. FT-IR is a promising, rapid, and low-cost method for the phenotypic classification of S. pneumoniae capsular serotypes. This methodology has significant implications for clinical and epidemiological practice, improving patient management, monitoring infection trends, and developing new vaccines.

Molecular characterization of the HMTp210 gene of Avibacterium paragallinarum and the proposition of a new genotyping method as alternative for classical serotyping

ABSTRACT Avibacterium paragallinarum (A. paragallinarum) is the aetiological agent of infectious coryza (IC) in chickens and characterized by acute respiratory distress and severe drop in egg production. Vaccination is important in the control of IC outbreaks and the efficacy of vaccination is dependent on A. paragallinarum serovars included in the vaccine. Classical serotyping of A. paragallinarum is laborious and hampered by poor availability of antigens and antisera. The haemagglutinin, important in classical serotyping, is encoded by the HMTp210 gene. HMTp210 gene analysis has been shown to have potential as alternative to classical serotyping. The aim of the present study was to further investigate the potential of sequence analyses of partial region 1 of the HMTp210 gene, the HMTp210 hypervariable region and the concatenated sequences of both fragments. For this analysis, 123 HMTp210 gene sequences (field isolates, A. paragallinarum serovar reference strains and vaccine strains) were included. Evaluation of serovar references and vaccine strains revealed a need for critical evaluation, especially within Page serovar B and C. Phylogenetic analysis of HMTp210 region 1 resulted in a separation of Page serovar A, B and C strains. Analysis of the HMTp210 HVR alone was not sufficient to discriminate all nine different Kume serovar references. The concatenated sequences of HMTp210 region 1 and HMTp210 HVR resulted in 14 clusters with a high correlation with Page serovar and with the nine currently known Kume serovars and is therefore proposed as a novel genotyping method that could be used as an alternative for classical serotyping of A. paragallinarum.

Genomic sequencing of fourteen Bacillus thuringiensis isolates: insights into geographic variation and phylogenetic implications

ObjectiveThis work was performed in support of a separate study investigating the activity of pesticidal proteins produced by Bacillus thuringiensis against the Asian citrus psyllid, Diaphorina citri. The fourteen Bacillus isolates chosen were selected from a large, geographically diverse collection that was characterized only by biochemical phenotype and morphology of the parasporal crystal, hence, for each isolate it was desired to determine the specific pesticidal proteins produced, assign each to a Bacillus cereus multilocus sequence type (ST), and predict their placement within the classical Bt serotyping system. In addition, phylogenetic distances between the isolates and Bacillus thuringiensis serovar type strains were determined by calculating digital DNA-DNA hybridization (dDDH) values among the isolates.ResultsBased on the assembled sequence data, the isolates were found to be likely representatives of the Bt serovars kurstaki (ST 8), pakistani (ST 550), toumanoffi (ST 240), israelensis (ST 16), thuringiensis (ST 10), entomocidus (ST 239), and finitimus (ST 171). In cases where multiple isolates occurred within a predicted serovar, pesticidal protein profiles were found to be identical, despite the geographic diversity of the isolates. As expected, the dDDH values calculated for pairwise comparisons of the isolates and their apparent corresponding Bt serovar type strains were quite high (> 98%), however dDDH comparisons of the isolates with other serovar type strains were often surprisingly low (< 70%) and suggest unrecognized taxa within Bt and the Bacillus cereus sensu lato.

Long-term surveillance of group B Streptococcus strains isolated from infection and colonization in pregnant women and newborns.

Introduction. Group B Streptococcus (GBS) remains the leading cause of bacterial neonatal infections worldwide, despite the spread of recommendations on vaginal screening and antibiotic prophylaxis.Hypothesis/Gap Statement. There is a need to evaluate the potential changes in GBS epidemiology over time following the introduction of such guidelines.Aim. Our aim was to perform a descriptive analysis of the epidemiological characteristics of GBS by conducting a long-term surveillance of strains isolated between 2000 and 2018, using molecular typing methods.Methodology. A total of 121 invasive strains, responsible for maternal infections (20 strains), fetal infections (8 strains) and neonatal infections (93 strains), were included in the study, representing all the invasive isolates during the period; in addition, 384 colonization strains isolated from vaginal or newborn samples were randomly selected. The 505 strains were characterized by capsular polysaccharide (CPS) type multiplex PCR assay and the clonal complex (CC) was assigned using a single nucleotide polymorphism PCR assay. Antibiotic susceptibility was also determined.Results. CPS types III (32.1 % of the strains), Ia (24.6 %) and V (19 %) were the most prevalent. The five main CCs observed were CC1 (26.3 % of the strains), CC17 (22.2 %), CC19 (16.2 %), CC23 (15.8 %) and CC10 (13.9 %). Neonatal invasive GBS diseases were predominantly due to CC17 isolates (46.3 % of the strains), which mainly express CPS type III (87.5 %), with a very high prevalence in late-onset diseases (76.2 %).Conclusion. Between 2000 and 2018, we observed a decrease in the proportion of CC1 strains, which mainly express CPS type V, and an increase in the proportion of CC23 strains, mainly expressing CPS type Ia. Conversely, there was no significant change in the proportion of strains resistant to macrolides, lincosamides or tetracyclines. The two molecular techniques used in our study provide almost as much information as classical serotyping and multilocus sequence typing, but are quicker, easy to perform, and avoid long sequencing and analysis steps.

Intravenous Infection of Small Ruminants Suggests a Goat-Restricted Host Tropism and Weak Humoral Immune Response for an Atypical Bluetongue Virus Isolate.

Bluetongue virus (BTV) is the etiologic agent of bluetongue (BT), a viral WOAH-listed disease affecting wild and domestic ruminants, primarily sheep. The outermost capsid protein VP2, encoded by S2, is the virion's most variable protein, and the ability of reference sera to neutralize an isolate has so far dictated the differentiation of 24 classical BTV serotypes. Since 2008, additional novel BTV serotypes, often referred to as "atypical" BTVs, have been documented and, currently, the full list includes 36 putative serotypes. In March 2015, a novel atypical BTV strain was detected in the blood of asymptomatic goats in Sardinia (Italy) and named BTV-X ITL2015. The strain re-emerged in the same region in 2021 (BTV-X ITL2021). In this study, we investigated the pathogenicity and kinetics of infection of BTV-X ITL2021 following subcutaneous and intravenous infection of small ruminants. We demonstrated that, in our experimental settings, BTV-X ITL2021 induced a long-lasting viraemia only when administered by the intravenous route in goats, though the animals remained healthy and, apparently, did not develop a neutralizing immune response. Sheep were shown to be refractory to the infection by either route. Our findings suggest a restricted host tropism of BTV-X and point out goats as reservoirs for this virus in the field.

A Walk through Gumboro Disease

Infectious bursal disease (IBD), caused by an Avibirnavirus, belonging to the family Birnaviridae, is an immunosuppressive disease that affects 3–6-week-old chickens, resulting in clinical or subclinical infection. Although clinical disease occurs in chickens, turkeys, ducks, guinea fowl, and ostriches can be also infected. IBD virus (IBDV) causes lymphoid depletion of the bursa, which is responsible for the severe depression of the humoral antibody response, primarily if this occurs within the first 2 weeks of life. IBD remains an issue in chicken meat production due to economic losses caused by the spread of variants or subtypes, resistant to the most common vaccines, responsible for a subclinical disease characterized by reduced growth performance and increased susceptibility to secondary infections. Very virulent strains of classical serotype 1 are also common in several countries and can cause severe disease with up to 90% mortality. This review mainly focuses on the immunosuppressive effect of the IBDV and potential vaccination strategies, capable of overcoming challenges associated with the optimal time for vaccination of offspring, which is dependent on maternal immunity and IBDV variant occurrence.

Identification of Five Serotypes of Enteropathogenic Escherichia coli from Diarrheic Calves and Healthy Cattle in Belgium and Comparative Genomics with Shigatoxigenic E. coli.

Simple SummaryEnteropathogenic Escherichia coli (EPEC) from cattle receive little attention, although some belong to the most notorious O serotypes of attaching/effacing Shigatoxigenic Escherichia coli (AE-STEC) responsible for the uremic and hemolytic syndrome in humans, such as O26. Nevertheless, the O serotypes and virulotypes of the large majority of bovine EPEC remain unidentified. This study aimed to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) by a polymerase chain reaction (PCR) among three collections of bovine EPEC from young diarrheic calves, healthy cattle at slaughterhouses, and healthy calves in dairy farms. The virulotypes and sequence types (STs) obtained after the whole genome sequencing of several O156, O177, and O182 bovine EPEC were closely related or identical to the virulotypes and STs of ten bovine and the human AE-STEC of the same O:H serotype. This study allows us to identify more EPEC O serotypes from cattle and to speculate on their evolution. Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (AE) lesions and cause non-bloody diarrhea in mammals. A minority of bovine EPEC belong to one of the ten classical serotypes of human and bovine AE-STEC. The purpose of this study was to identify five non-classical O serotypes (O123/186, O156, O177, O182, and O183) among bovine EPEC and to characterize their virulence repertoires by whole genome sequencing. Around 40% of the 307 EPEC from 307 diarrheic calves, 368 EPEC from 47 healthy cattle, and 131 EPEC from 36 healthy calves in dairy farms were analyzed. Serotype O177 was the most frequent among EPEC from diarrheic and healthy calves, while the O156 was the most frequent in healthy cattle. The genomic analysis identified different H serotypes, MLSTypes, and/or eae gene subtypes among the O156 and O177 EPEC, while the O182 was homogeneous. The virulence gene profiles of bovine EPEC were closely related to each other and to the profiles of ten bovine and human AE-STEC. These results emphasize the need for additional studies to identify more O:H serotypes of bovine EPEC and to elucidate their origin and evolution of EPEC with regard to AE-STEC belonging to the same O:H serotypes.

Bluetongue Virus Infection of Goats: Re-Emerged European Serotype 8 vs. Two Atypical Serotypes.

In recent years, numerous atypical Bluetongue virus (BTV) strains have been discovered all around the world. Atypical BTV strains are phylogenetically distinct from the classical BTV serotypes 1–24 and differ in terms of several biological features. For the first time, the atypical strains BTV-25-GER2018 and BTV-33-MNG3/2016 as well as the re-emerged classical strain BTV-8-GER2018 were evaluated comparatively in a pathogenesis study in goats—the natural host of atypical BTV. A substantial number of in-contact animals were included in this study to detect potential contact transmissions of the virus. After infection, EDTA blood, ocular, nasal and oral swab samples as well as serum were collected regularly and were used for virological and serological analyses, respectively. Our study showed differences in the immunological reaction between the two atypical BTV strains (no group-specific antibody detection) and the classical BTV strain BTV-8-GER2018 (group-specific antibody detection). Furthermore, we observed an increase in the total WBC count (neutrophils and lymphocytes) in goats infected with the atypical BTV strains. No horizontal transmission was seen for all three strains. Our study suggests that the atypical BTVs used in the trial differ from classical BTVs in their immunopathogenesis. However, no evidence of direct contact transmission was found.

Detection of multiple human astroviruses in sewage by next generation sequencing

Human astrovirus (HAstV) composes of classic HAstV serotypes 1–8 and recently discovered novel HAstV-MLB and HAstV-VA strains. A number of studies have demonstrated that wastewater analysis is an effective approach to understand the prevalence and diversity of enteric viruses in local population. However, a comprehensive analysis of classic and novel HAstVs in sewage is still lacking. In this study, sewage samples were collected monthly from Jinan, China during 2018–2019. Quantification of HAstV genomes was performed by real-time quantitative PCR. Different from previous studies which focused on partial ORF1b or ORF2 gene, complete ORF2 region of HAstV was amplified from sewage concentrates, and amplicons were subjected to next generation sequencing (NGS) and genetic analysis. This methodology allowed detection of 18 astroviruses, of which 7 (HAstV-1, -2, -4, -5, VA1, VA2, and VA3) were detected in all sewage samples. A new strain VA6 mapped to the HMO clade was identified in 20.8% of samples, with 82.4%–83.3% nucleotide identities to the closest strain VA5. The viral load of classic, MLB and VA clades in sewage samples ranged from 3.7 × 104 to 4.6 × 107, 3.4 × 104 to 3.9 × 106, and 3.3 × 104 to 4.1 × 106 copies per liter, respectively. Phylogenetic analysis based on complete ORF2 region reflected local HAstVs within each genotype constituted multiple co-circulating lineages. Existence of several new lineages composed exclusively or predominantly of Chinese sequences was observed as well. These results demonstrate sewage contains astroviruses with considerable high diversities. NGS based environmental surveillance greatly improves the understanding of HAstV circulation and should be encouraged.

Changing epidemiology of human enteroviruses (HEV) in a hand, foot and mouth disease outbreak in Vellore, south India

PurposeHand Foot and mouth disease (HFMD) is a major childhood exanthematous disease causing outbreaks that have become a major public health threat in recent years. In Vellore district of Tamil Nadu, south India, occasional outbreaks are common among the paediatric age group, most commonly in those under 5years of age (U5s). CoxsackieA6, A4, A5, A9, A10, B2 and B5 are the common serotypes causing outbreaks. This study aimed to identify the molecular serotype of the causative agent, co-circulating in this region. MethodsAdapting the WHO case definition, cases during an HFMD outbreak between October and December 2017, were identified by a clinical criterion of fever, mouth ulcers and rash in the extremities. Vesicle fluid from these lesions were collected in viral transport medium and transported cold to the Clinical Virology laboratory of a tertiary care hospital in Vellore. Identification of the causative agent was undertaken by two real time PCRs (EV1 and EV2) followed by sequencing the VP1–2C region and constructing a phylogenetic tree. ResultsAmong the 31 HFMD patients included in this study, 23 (74.2%) were U5s, 3 (9.7%) were between 6 and 15 years and the remaining 5 (16.1%) were adolescents (>15 ​yrs). The outbreak ran a mild clinical course, with 22(71%) patients having fever as a prodromal symptom. Papulovesicular lesions characteristic of HFMD were present on all 31 (100%) patients’ palms and soles, buttocks of 19 (61.3%), oral mucosa of 12 (38.7%), and all over the body in 4 (12.9%) patients. Coxsackie A6(75%) and Coxsackie A16(25%) were the pathogens associated with this outbreak. ConclusionsChanging epidemiology of HFMD was seen in this outbreak since; other serotypes apart from the classical Coxsackievirus serotypes causing HFMD outbreak were also found co-circulating. EV1 PCR was a better screening assay than EV2 PCR in this region. Continued surveillance and molecular serotyping are necessary for HFMD outbreaks in any region.

Astrovirus-induced epithelial-mesenchymal transition via activated TGF-β increases viral replication.

Human astroviruses (HAstV), positive sense single-stranded RNA viruses, are one of the leading causes of diarrhea worldwide. Despite their high prevalence, the cellular mechanisms of astrovirus pathogenesis remain ill-defined. Previous studies showed HAstV increased epithelial barrier permeability by causing a re-localization of the tight junction protein, occludin. In these studies, we demonstrate that HAstV replication induces epithelial-mesenchymal transition (EMT), by upregulating the transcription of EMT-related genes within 8 hours post-infection (hpi), followed by the loss of cell-cell contacts and disruption of polarity by 24 hpi. While multiple classical HAstV serotypes, including clinical isolates, induce EMT, the non-classical genotype HAstV-VA1 and two strains of reovirus are incapable of inducing EMT. Unlike the re-localization of tight junction proteins, HAstV-induced EMT requires productive replication and is dependent transforming growth factor-β (TGF-β) activity. Finally, inhibiting TGF-β signaling and EMT reduces viral replication, highlighting its importance in the viral life cycle. This finding puts classical strains of HAstV-1 in an exclusive group of non-oncogenic viruses triggering EMT.

Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan.

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

Six decades of infectious bursal disease in poultry: The journey so far and challenges ahead

&#x0D; &#x0D; &#x0D; Despite six decades of concerted efforts, Infectious bursal disease (IBD) still remains a major threat to the poultry industry worldwide. Most importantly, the emergence of variant and very virulent strains of infectious bursal disease virus (IBDV) has dramatically changed the epidemiology of the disease, thus resulting in the renewed efforts in the search for effective control measures. Currently, live attenuated, inactivated, and immune-complex vaccines are among the immune-therapeutic approaches employed for the control of IBD in the field alongside adequate biosecurity, albeit with various degrees of success and limitations. Progress in genetic engineering has allowed the generation of reverse genetic IBDV mutants, recombinant live viral vectors expressing the IBDV VP2 immunodominant protein, intra-serotypic recombinant IBDV viral-like particle co-expressing the outer capsid protein structures derived from 2 or more serotype 1 strains or the incorporation of either VP2 or VP2-4-3 polyprotein sequences alongside molecular adjuvants that can be used as IBD vaccine candidates to elicit an immune response. However, despite these advances, outbreaks are still reported even in flocks that have up to date vaccination records and somewhat excellent management practices. This paper reviews aspect of genetic characteristics of IBDV and reflects on the progress and future challenges in providing effective IBD vaccine to achieve effective control of both classical and very-virulent IBDV serotypes that constitute a major devastation to poultry production and health.&#x0D; &#x0D; &#x0D;

Prospective Study in Children with Complicated Urinary Tract Infection Treated with Autologous Bacterial Lysates.

Antimicrobial bacteria resistance is an important problem in children with recurrent urinary tract infections (rUTI), thus it is crucial to search for alternative therapies. Autologous bacterial lysates (ABL) may be a potential treatment for rUTI. Twenty-seven children with rUTI were evaluated for one year, urine and stool cultures were performed, 10 colonies of each culture were selected and those identified as Escherichia coli were characterized by serology. For patients who presented ≥105 UFC/mL, an ABL was manufactured and administered orally (1 mL/day) for a month. Twelve children were monitored for ≥1-year, 218 urine and 11 stool samples were analyzed. E. coli (80.5%) was the main bacteria isolated from urine and feces (72%). E. coli of classical urinary serotypes (UPEC), O25:H4, O75:HNM, and O9:HNM were identified in patients with persistent urinary infection (pUTI). In 54% of patients treated with ABL, the absence of bacteria was observed in urine samples after 3 months of treatment, 42% of these remained without UTI between 10–12 months. It was observed that the use of ABL controlled the infection for almost 1 year in more than 60% of the children. We consider it necessary to develop a polyvalent immunogen for the treatment and control of rUTI.

Human Astrovirus 1-8 Seroprevalence Evaluation in a United States Adult Population.

Human astroviruses are an important cause of viral gastroenteritis globally, yet few studies have investigated the serostatus of adults to establish rates of previous infection. Here, we applied biolayer interferometry immunosorbent assay (BLI-ISA), a recently developed serosurveillance technique, to measure the presence of blood plasma IgG antibodies directed towards the human astrovirus capsid spikes from serotypes 1–8 in a cross-sectional sample of a United States adult population. The seroprevalence rates of IgG antibodies were 73% for human astrovirus serotype 1, 62% for serotype 3, 52% for serotype 4, 29% for serotype 5, 27% for serotype 8, 22% for serotype 2, 8% for serotype 6, and 8% for serotype 7. Notably, seroprevalence rates for capsid spike antigens correlate with neutralizing antibody rates determined previously. This work is the first seroprevalence study evaluating all eight classical human astrovirus serotypes.

Development of a Genoserotyping Method for Salmonella Infantis Detection on the Basis of Pangenome Analysis

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

Incidence of Salmonella serovars isolated from commercial animal feed mills in the United States and serovar identification using CRISPR analysis.

In this study, we sought to determine the incidence and diversity of Salmonella in a broad collection of commercial animal feeds collected from animal feed mills across the United States over an 11-month period and utilize CRISPR analysis to identify individual serovars. Over two independent trials, 387 feed samples from 135 different animal feed mills in the United States were screened for Salmonella. A total of 6·2% (24/387) of samples were contaminated with Salmonella, which is concordant with similar studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-typing was used to serotype Salmonella isolates, and serovars Infantis and Tennessee were the most common. Serogroups O:4 and O:7 were enriched in the feed samples, suggesting that these serogroups are better adapted to surviving in low moisture animal feeds. The study supports the utility of CRISPR to determine serovar type since most of the serovars identified in this study have been also isolated and identified in earlier studies using more classical serotyping methods. This work contributes to a growing body of literature concerning the Salmonella prevalence in animal feeds and highlights the need to effectively mitigate pathogens in livestock and poultry feed.

Evolutionary Analysis of Infectious Bronchitis Virus Reveals Marked Genetic Diversity and Recombination Events.

In the last 5 years, frequent outbreaks of infectious bronchitis virus (IBV) are observed in both broiler and layer chicken flocks in the Kingdom of Saudi Arabia (KSA) in spite of extensive usage of vaccines. The IBV is a widespread avian coronavirus affecting both vaccinated and unvaccinated chicken flocks and is attributed to significant economic losses, around the globe. In the present study, 58 (n = 58) samples were collected from four different commercial poultry flocks from 8 KSA districts during 2019. A total of nine positive isolates (9/58; 15.5%), based on real-time reverse transcriptase PCR targeting nucleocapsid (N) gene, were used for further genetic characterization and evolutionary analysis. Genetic characterization of the partial spike (S1) gene revealed the clustering of the reported isolates into three different genotypes, whereas four additional isolates were grouped within 4/91 genotype, two isolates within IS/885 genotype, one isolate was closely related to IS/1494/06, and two isolates were grouped within classic serotype (vaccine-like strains). Phylodynamic revealed clustering of four isolated viruses within GI-13 lineage, three isolates within GI-23 lineage, and two isolates within GI-1 lineage. Results indicate that there are high evolutionary distances between the newly identified IBV strains in this study and the commercially used vaccines (GI-1), suggesting that IBV strains circulating in the KSA are under constant evolutionary pressures. Selective pressure biostatistics analyses consistently demonstrate the presence of a higher positive score which highlights the role of natural selection, a mechanism of virus evolution on sites located on the protein surface, within or nearby domains involved in viral attachment or related functions. Recombination analysis revealed emergence of two isolates through recombination events resulting in new recombinant viruses. Taken together, these finding demonstrate the genetic and evolutionary insights into the currently circulating IBV genotypes in KSA, which could help to better understand the origin, spread, and evolution of infectious bronchitis viruses, and to ascertain the importance of disease monitoring as well as re-evaluation for the currently used vaccines and vaccination programs.